Inhibition of viral infection using antigen-binding proteins

ABSTRACT

A method of inhibiting viral infection using a monovalent antigen binding protein comprising a single domain binding unit capable of binding to a virus is described. Preferably the protein is a heavy chain variable domain derived from an immunoglobulin naturally devoid of light chains. Food, pharmaceutical and cosmetic products comprising such proteins are also described together with a method for selecting inhibiting proteins from a large population of mainly containing non-inhibiting, but infectious agent binding fragments.

FIELD OF THE INVENTION

[0001] The present invention relates to the use of antigen bindingproteins in a method of inhibiting the infectivity of viruses or otherinfectious agents, products and compositions comprising such proteinsand methods for identifying and/or selecting antigen binding proteinscapable of exhibiting such activity. In particular, the inventionrelates to a method for inhibiting viral infection using a monovalentantigen binding protein comprising a variable domain of a heavy chainderived from an immunoglobulin devoid of light chains, capable ofbinding to a virus.

BACKGROUND OF THE INVENTION

[0002] Antibodies are protein molecules belonging to a group ofimmunoglobulins generated by the immune system in response to anantigen. The structure of most antibody molecules is based on a unitcomprising four polypeptides, two identical heavy chains and twoidentical light chains, which are covalently linked together bydisulphide bonds. Each of these chains is folded in discrete domains.The C-terminal regions of both heavy and light chains are conserved insequence and are called the constant regions, comprising one or moreso-called C-domains. The N-terminal regions of the heavy and lightchains, also known as V-domains, are variable in sequence and determinethe specificity of the antibody. The regions in the variable domains ofthe light and heavy chains (V_(L) and V_(H) respectively) responsiblefor antigen binding activity are known as the hypervariable orcomplementarity determining regions (CDR).

[0003] Immunoglobulins capable of exhibiting the functional propertiesof the four-chain immunoglobulins described above but which comprise twoheavy polypeptide chains and which furthermore are devoid of lightpolypeptide chains have been described (Wo 94/04678, Casterman et al,1994). Fragments corresponding to isolated V_(H) domains (hereinafterVHH) are also disclosed. Methods for the preparation of such antibodiesor fragments thereof on a large scale comprising transforming a mould oryeast with an exoressible DNA sequence encoding the antibody or fragmentare described in patent application WO 94/25591 (Unilever).

[0004] The immunoglobulins described in WO 94/04673, which may beisolated from the serum of Camelids, do not rely upon the association ofheavy and light chain variable domains for the formation of theantigen-binding site but instead the heavy polypeptide chains alonenaturally form the complete antigen binding site. These immunoglobulins,hereinafter referred to as “heavy-chain immunoglobulins” are thus quitedistinct from the heavy chains obtained by the degradation of common(four-chain) immunoglobulins or by direct cloning which contribute partonly of the antigen-binding site and require a light chain partner forantigen-binding, thus forming a complete antigen binding site.

[0005] Antibodies or fragments thereof, have found application in avariety of uses where the specific nature of the antibody-antigeninteraction can be used to advantage. These include such uses asdiagnosis, therapy, immunoassays and purification processes. The use ofantibodies, or fragments thereof, in inhibiting viral infection hasreceived attention, for instance during active immunisation withinactivated virus preparations or viral antigens produced in recombinantcells or during passive immunisation by the administration ofneutralising antibodies.

[0006] It has been reported in the literature that monovalent Fabantibody fragments can neutralise viruses. Cheung et al (1992), Journalof Virology, 66, 6714-6720, describe the production of the Fab domain ofa rabies virus-neutralising antibody MAb-57 and further demonstrate thatthis monovalent fragment itself has virus-neutralising activity. Otherpublications also report the capability of human Fab monovalent antibodyfragments to neutralise or inhibit viral activity (see for example,Williamson et al (1993), Proc. Natl. Acad. Sci. USA, 90, 4141-4145).Such methods are not suitable for wide scale industrial application asthe cost of producing such classical antibody fragments renders theprocesses economically unfeasible.

[0007] An alternative approach to inhibiting viral replication usingantibodies which has been described in the literature is to selectantibodies to target enzymes produced by the virus. Martin et al,Protein Engineering, 10(5), 607-614 (1997) describes the use of acamelisedf VH antibody fragment to inhibit hepatitis C virus MS3protease, thereby preventing cleavage of the viral poly-proteinprecursor.

[0008] Another industrial application in which economically viablesolutions to the problem of viral infection are sought is the field offermentation processing, particularly food processing.

[0009] Lactic acid bacteria (LAB: Lactococci and Lactobacilli) play animportant role in food fermentation processes such as the production ofcheese or yoghurt. Often such fermentations are hampered by thesensitivity of the bacteria towards viruses, known as bacteriophage,which build up in these, often not aseptically performed, processes. Aphage infection causes the LAB cells to lyse; during prolongedfermentations phage resistant cell populations can evolve, but thisdelay affects the production capacity severely, and the disturbedprocess yields a product of low quality. Sometimes the process has to bestopped prematurely, with complete loss of the batch of milk.

[0010] To date, the phage problem has mainly been approached by takingspecial precautions with respect to hygiene at the production facility,but this causes additional time delays. Another solution which has beenproposed is the use of resistant LAB strains, but the regular appearanceof adapted forms of bacteriophage forces the strains used to be changedfrom time to time in a procedure known as culture rotation. This has thedisadvantage age of requiring labour intensive monitoring of theproduction facilities and medium for the presence of phage and requiresthe availability of several sets of cultures with the same functionalattributes, differing only in phage sensitivity. There therefore remainsconsiderable commercial interest in the further development of methodsfor combating LAB phage infection.

[0011] One method, proposed by Geller et al (1998), J. Dairy Sci., 81,895-900, involves the use of colostrum from cows immunised withlactococcal phage as a source of phage-neutralising (polyclonal)antibodies to prevent lytic infection of Lactococcus lactis infermentations of phage-contaminated milk. This method does provide acommercially viable solution to the problem, however. Not only is itextremely economically unattractive to produce antibodies in this waybut furthermore, the addition of colostrum to milk does not haveregulatory approval.

[0012] An alternative approach, which makes use of multivalent,multispecific antigen binding proteins comprising a polypeptidecomprising in series two or more single domain binding units, preferablyvariable domains of a heavy chain derived from an immunoglobulinnaturally devoid of light chains, to reduce the infectivity of LABphages by cross-linking or agglutination is exemplified in theApplicant's co-pending patent application number PCT/EP98/06991, filedOct. 26, 1998.

[0013] There remains a continuing need for the development of improvedmethods of inhibiting or neutralising viral infection. In particular,there remains continuing interest in development of methods which can beapplied economically on a scale appropriate for industrial use.

SUMMARY OF THE INVENTION

[0014] Accordingly, the invention provides in one aspect a method ofinhibiting viral infection using a monovalent antigen binding proteincomprising a single variable domain binding unit, or a functionalequivalent thereof, capable of binding to a virus.

[0015] In another aspect the invention provides the use of a monovalentantigen binding protein comprising a single variable domain binding unitor a functional equivalent thereof capable of binding to a virus ininhibiting viral infection.

[0016] The invention also provides the use of a monovalent antigenbinding protein comprising a single variable domain binding unit or afunctional equivalent thereof capable of binding to a virus in thepreparation of a medicament for inhibiting viral infection.

[0017] Also provided are monovalent antigen binding proteins comprisinga single variable domain binding unit capable of binding to a virus,nucleotide sequences encoding such proteins, cloning end expressionvectors comprising such nucleotide sequences, host cells transformedwith vectors comprising such nucleotide sequences, and food, cosmeticand pharmaceutical products comprising such proteins.

[0018] In a further aspect, the invention provides a method forselecting an antigen binding protein capable of inhibiting viralinfection of a host cell comprising the steps of:

[0019] i) complexing an antigen binding protein with a target virus,

[0020] ii) exposing the antigen binding protein-virus complex of step(i) to an excess of host cells,

[0021] iii) removing the host cells and any associated antigen bindingprotein-virus complex,

[0022] iv) capturing antigen binding protein-virus complex not taken upby the host cells in step (ii) with virus specific ligands to separatevirus specific antigen binding proteins from non-binding proteins.

[0023] The invention also provides a method for identifying an antigenbinding protein capable of inhibiting bacteriophage infection of alactic acid bacterial cell host comprising the steps of:

[0024] i) culturing of bacterial host cells in the presence of antigenbinding protein and bacteriophage,

[0025] ii) assaying said culture for active cell growth manifest in achange in pH of the culture growth medium.

[0026] As used herein, a single variable domain binding unit means animmunoglobulin variable domain or a functional equivalent thereof whichforms a complete antigen binding site. This may be derived from naturalsources or synthetically produced. The terms ‘immunoglobuiin’ and‘antibody’ are used synonymously throughout the specification, unlessindicated otherwise.

[0027] A ‘functional equivalent’ of an imunoglobulin variable domain isany homogolous protein molecule which has similar binding specificity. Afunctional equivalent may be characterised by an insertion, deletion orsubstitution of one or more amino acid residues in the sequence of theimmunoglobulin variable domain. Suitably, the amino acid sequence of thefunctional equivalent has at least 60% similarity, preferably at least80%, more preferably at least 90% similarity to the amino acid sequenceof the immunoglobulin variable domain.

[0028] Inhibition of viral infection includes but is not limited toinhibition of infection by blocking essential sites on the viralparticle, such as the receptor binding protein of the virus by which thevirus attaches to the host cell during the first step of infection.Inhibition may be total or partial. The terms ‘inhibit’ and ‘neutralise’are used synonomously herein.

[0029] The term ‘virus’ includes within its scope viruses, which infectbacterial host cells, known as bacteriophages. Binding to a virusincludes binding to one or more molecules located at the surface of thevirus particle.

[0030] The present invention may be more fully understood with referenceto the following description when read together with the accompanyingdrawings in which:

[0031]FIG. 1 shows the efficiency of the selected monovalent VHHfragment VHH#1 in neutralising Lactococcus lactis bacteriophage P2 asmeasured by plaque titration.

[0032]FIG. 2 shows the prevention of phage infection by VHH fragmentVHH#1 in an acidification experiment with a small scale culture of milk.

[0033]FIG. 3 shows the determination of the valency of the antibodyfragments used in this study with an ELISA based method.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The invention is based on the finding that a monovalent 2 antigenbinding protein comprising a single variable domain binding unit capableof binding to a virus can advantageously be used to inhibit infection ofa host cell by the virus. High production levels and ease of downstreamprocessing permits such antigen binding proteins to be applied inindustrial processes and products economically and efficiently.

[0035] As discussed above, antibody based methods for inhibiting viralinfection which have previously been described in the literature haverelied either on cross-linking mediated via multivalent constructs orhave made use of larger fragments derived from ‘classical’ antibodies,such as Fab fragments, to block the receptor binding protein of thevirus, hence inhibiting its ability to infect the host cell.

[0036] Surprisingly, the present inventors have found that much smallermonovalent antigen binding proteins comprising a single variable domainare effective in inhibiting viral infection. This would not have beenpredicted from the prior art teaching as the smaller size of theseproteins might have been expected to have rendered them less effectivein hindering binding of the virus to the host cell. Moreover, it wouldbe expected that multivalent antigen binding proteins would be moreeffective due to the agglutination of infectious particles. To date,only the recognition of simple protein antigens with such bindingproteins has beer reported. There has been no suggestion that complexsystems such as viruses could be detected and inhibited using singledomain binding units.

[0037] The invention is applicable to the use of any immunoglobulinvariable domain, which forms a complete antigen binding site. Theimmunoglobulin may be derived from natural sources or syntheticallyproduced. Preferably, the invention relates to the use of heavy chainvariable domains derived from an immunoglobulin devoid of light chains,most suitably from an immunoglobulin naturally devoid of light chainssuch as are obtainable from lymphoid cells, especially peripheral bloodlymphocytes, bone marrow cells or spleen cells derived from Camelids asdescribed in WO 94/04678 (Casterman et al) discussed above.

[0038] It will be appreciated that heavy chain variable domains derivedfrom other immunoglobulins modified (‘camelised’) to enable them tofunction as monovalent binding domains in the same way as the heavychain variable domains derived from Camelids may also suitably be usedaccording to the invention.

[0039] An advantage of using single domain binding units which are heavychain variable domains derived from Camelids is that they can readilyand conveniently be produced economically on a large scale, for exampleusing a transformed lower eukaryotic host as described in Wo 94/25591(Unilever). A major advantage of the described production system is thelow degree of impurities present in the secreted fraction, therebyenabling simple down stream processing procedures for purification. Afurther advantage, particularly when applications in food processing arecontemplated, is that such heavy chain variable domains are extremelyheat stable, allowing pasteurisation or other heat treatments withoutloss of antigen binding capacity.

[0040] The invention is applicable to both prokaryotic and eukaryotichost cells. For therapy of humans or animals, target viruses of interestinclude pathogenic viruses such as those which belong to the family ofHuman Immunodeficiency Viruses. Other viral infections to which theinvention is applicable include such food-born viruses as Hepatitisviruses (especially Hepatitis A virus), Rotavirus and the small roundStructured viruses (SRSV), such as Norwalk virus (see Food Science andTechnology Today, II(1), 49-51, 1997). In the area of production crops,viruses pathogenic to plants such as Citrus tristeza virus (CTV),Tobacco mosaic virus (TMV), Potato virus Y (PVY), Lettuce necroticyellows virus (LNYV), Tomato spotted wilt virus (TSWV), Clover woundtumour virus (CWTV), Cauliflower mosaic virus (CaMV), Cowpea mosaicvirus (CPMV), Soil-borne wheat furiovirus (SBWMV), Wheat yellow mosaicbymovirus (WYMV) and Wheat spindle streak mosaic virus (WSSMV) areimportant targets for neutralisation or inhibition.

[0041] Single domain binding units such as heavy chain variable domainsrecognising plant viruses can be cloned and expressed in plants using,methods equivalent to conventional cloning and expression of (modified)viral proteins, to protect these plants viruses. By using theappropriate targeting signals known in the art, the expression andtranslocation of the VHH's can be regulated in such a way that organelleor in the extracellular matrix.

[0042] The invention is of particular use in industrial fermentationprocesses, for example neutralising or inhibiting the infectivity oflactococcal bacteriophage, thereby preventing lytic infection ofLactococcus lactis. By inhibiting the infectivity of lactococcalbacteriophages, the invention affords the possibility of avoiding havingto adopt the various cost affecting measures described above. Theantigen binding proteins can be used in a cleaning product, whichremoves phage present in the production system. Alternatively, they canbe added to milk contaminated with bacteriophage, as is shown in Example3 below, which can be fermented to a high quality product without anydelay in the production. The standard addition of such antigen bindingproteins to milk would be one way in which it might be possible toabolish monitoring for the presence of phage.

[0043] Lactic acid bacteria play an important part in fermentation ofmany other food products in addition to dairy products. It will beappreciated that the invention is not restricted to use in inhibitingLAB phage infection in dairy fermentation processes but extends also touse in any process which makes use of lactic acid bacteria fermentation.Suitable fermented food products and the associated lactic acid bacteriaare listed in tables 1a-1b below (see Biotechnology, Vol 5, Chapter1-8). TABLE 1a Main functional lactic acid bacteria in EuropeanFermented Foods MAIN LACTIC ACID PRODUCT NAME SUBSTRATE BACTERIA BakedGoods Wheat Lactobacillus plantarum acidophilus delbrueckii brevisbuchneri fermentum s. francisco Wine & Brandy Grapes Leuconostoe gracileoenos Lactobacillus plantarum casei fructivorans hilgardii brevisPediococcus cerevisiae Cheese & Dairy Milk Brevibacterium linensProducts Lactococcus lactis cremoris Lactobacillus casei helveticusbulgaricus plantarum Leuconostoc cremoris Pediococcus acidilacticipentosaceus Sreptococcue thermophilus Enterococcus faecium FermentedCabbage & Lactobacillus brevis vegetables/fruits Cucumbers plantarumLeuconostoc mesenteroides Pediococcus cerevisiae Olives Lactobacillusplantarum paracasei brevis delbrueckii Streptoccoccus sp. Pediococcussp. Leuconostoc sp. Sausages Meat Lactobacullus curvatus lactisplantarum sake Pediococcus acidilactici pentocaceus Micrococcuscaseolyticus

[0044] TABLE 1b Main functional lactic acid bacteria in IndigenousFermented Foods PRODUCT NAME MAIN LACTIC ACID (Country) SUBSTRATESBACTERIA Banku (Ghana) Maize, cassava Lactic acid bacteria Burukutu(Nigeria) Sorghum, cassava Lactic acid bacteria Busa (Egypt) Rice,millet Lactobacillus sp. Dawadawa (Nigeria) Locust bean Lactic acidbacteria Dosai (India) Black gram and rice Lauconostoc mesenteroidesHamanatto (Japan) Whole soybean, Streptococcus sp. wheat flourPediococcus sp. Idli (India) Rice, black gram Leuconostoc mesenteroidesKecap (Indonesia) Soybean, wheat Lactobacillus sp. Kimchi (Korea)Vegetables Lactic acid bacteria (seafood, nuts) Kishk (Egypt) Wheat,milk Lactic acid bacteria Mshewu (S. Africa) Maize Lactobacillusdelbrueckii Miso (China, Japan) Rice and soybean Lactobacillus sp. Riceand cereals Lactobacillus sp. Ogi (Nigeria) Maize Lactic acid bacteriaPuto (Philippines) Rice Lactic acid bacteria Sorghum beer Sorghum, maizeLactic acid bacteria (S. Africa) Soybean milk (Asia) Soybean Lactic acidbacteria Soy sauce (Asia) Soybean and wheat Lactobacillus sp.Pediococcus sp. Tarhana (Turkey) Wheat and milk Lactic acid bacteria

[0045] The invention further provides nucleotide sequences coding forthe monovalent antigen binding proteins capable of inhibiting thecommonly occurring lactococcus bacteriophage P2. The inhibiting bindingdomains were identified with a high-throughput screening assay, whichallows the discrimination of inhibiting from non-inhibiting bindingproteins. The involved binding site on phage P2 was characterised byelectron microscopy with binding domain fragments conjugated to goldparticles. In addition, the cross-reactivity against members of the samefamily and of other families of bacteriophage was analysed in moredetail.

[0046] Particular heavy chain variable domains (referred to hereinafteras VHH fragments) of use according to the invention in inhibitinglactococcus bacteriophage P2 comprise the sequences: VHH#1 QVQLQESGGGLVQAGGSLRL SCTASRRTGS NWCMGWFRQL AGKEPELVVA LNFDYDMTYY (SEQ. ID NO. 1)ADSVKGRFTV SRDSGKNTVY LQMNSLKPED TAIYYCAARS GGFSSNRELY DGWGQGTQVT VSSVHH#2 QVQLQESGGG LVQAGGSLRL SCTASPRTGS NWSMGWFRQL AGKEREFVVA LNLDYDIPYY(SEQ. ID NO. 2) ADSVKGRFTV STDSGKNTVY LQMNSLKPED TAIYFCAARS GGFSSNRTYYDYWGQGTQVT VSS VHH#3 QVQLQQSGGG LVQRGGSLRL SGTASRRTGS NWSMGWFRQFAGKEPDLLVA LNLDYDVPYY (SEQ. ID NO. 3) ADSVKGRFTV SGDSGKNTVY LQMNNLKPEDTAIYYCAARS GGFSSNRALY DGWGQGTQVT VSS

[0047] The invention also provides host cells and expression vectorsenabling high level production and secretion of the binding proteins.

[0048] Heavy chain variable domains derived from an immunoglobulinnaturally devoid of light chains having a determined antigen specificitymay conveniently be obtained by screening expression libraries of clonedfragments of genes encoding Camelid immunoglobulins generated usingconventional techniques, as described, for example, in WO 94/04678 andExample 1. Suitable methods to enrich for binding domains recognisingthe infectious agent, thereby limiting the numbers of clones which haveto be screened for the identification of inhibiting fragments are yeastdisplay (WO 94/01.57 from Unilever) or phage display.

[0049] A preferred method to enrich for inhibiting binding domainsapplicable to the single variable domain binding units described herein,is based on the removal of clones that expose non-inhibiting bindingdomains, through capture of a complex of the binding domain and theinfectious agent of interest by host cells via a receptor protein towhich the non-inhibited infectious agent can bind.

[0050] Viral infection inhibiting antigen binding proteins according tothe invention may be prepared by transforming a host by incorporating agene encoding the polypeptide as set forth above and expressing saidgene in said host.

[0051] Suitably the host or hosts may be selected from prokaryoticbacteria, such as Gram-negative bacteria, for example E. coli, andGram-positive bacteria, for example B. subtilis and in particular lacticacid bacteria, lower eukaryotes such as yeasts, for example belonging tothe genera Saccharomyces, Kluyveromyces, Hansenula or Pichia, or mouldssuch as those belonging to the genera Aspergillus or Trichoderma.

[0052] Preferred hosts for use in connection with the present inventionare the lower eukaryotic moulds and yeasts, and in particular the lacticacid bacteria, which can be directly used for the fermentation of milk.

[0053] Techniques for synthesising genes, incorporating them into hostsand expressing genes in hosts are well known in the art and the skilledperson would readily be able to put the invention into effect usingcommon general knowledge.

[0054] Proteins for use according to the invention may be recovered andpurified using conventional techniques such as affinity chromatography,ion exchange chromatography or gel filtration chromatography.

[0055] The binding activity of the binding proteins according to theinvention may conveniently be measured by standard techniques known inthe art such as enzyme-linked immunoadsorbant assay (ELISA), radioimmuneassay (RIA) or by using biosensors. The inhibiting capacity may beexamined by the inhibition of plaque formation of phage and viruses, orby a method, which reveals continued cell growth as a measure forresistance against infection. In the case of lactococcus bacteriophage,the high throughput screening assay described in this application, or inan acidification experiment by the fermentation of milk is particularlyapplicable.

[0056] Antigen binding proteins capable of binding to a virus accordingto the present invention may conveniently be added to food or cosmeticcompositions by methods conventional in toe art to give products whichare protected against infection by the particular virus. Alternatively,the antigen binding proteins according to the invention may beformulated into pharmaceutical compositions with pharmaceuticallyacceptable carriers and/or excipients and optionally otherpharmaceutically or cosmetically active ingredients using techniqueswell known in the art.

[0057] The following examples are provided by way of illustration only.Techniques used for the manipulation and analysis of nucleic acidmaterials were performed as described in Sambrook et al, MolecularCloning, Cold Spring Harbor Press, New York, 2nd Ed. (1989), unlessotherwise indicated. Phages were isolated and propagated according tothe methods described by Moineau et al, Canadian Journal of Microbiology38/9, 875-882 (1992).

[0058] V_(H)H denotes heavy chain variable domain of heavy chainantibodies.

[0059] Restriction sites are underlined.

EXAMPLES Example 1 Induction of a Humoral Immune Response in Llama

[0060] A male llama was immunised with bacteriophage P2 of Lactococcuslactis in oil emulsion (1:9 V/V, antigen in water: Specol (Bokhout et al(1981), Immunol. Immunopath., 2, 491-500; Bokhout et al (1986), Infect.Dis., 161-168) subcutaneously and intramuscularly. Per immunisation site0.75-1.5 ml water in oil emulsion was infected containing 200 μg phageprotein (approx. 6*10¹³ pfu). Immunisations were performed according tothe following time schedule: the second immunisation was performed threeweeks after the first injection, and the third immunisation two weeksafter the second one. The immune response was followed by titration ofserum samples in ELISA with bacteriophage immobilised on Nunc maxi-sorbplates (coat solution 1010 pfu/ml diluted in phosphate buffered saline).After incubation with serum, the bound llama antibodies were detectedwith polyclonal rabbit-anti-llama antiserum (obtained via immunisingrabbits with llama immunoglobulines purified via ProtA and ProtGcolumns; ID-DLO) and swine-anti-rabbit immunoglobulines (DAIO)conjugated to horse radish peroxidase. Finally the peroxidaseenzyme-activity was determined with tetramethylbenzidine andureaperoxide as substrate and, after termination of the reaction byadding H₂SO₄, the optical density was measured at 450 nm.

Example 2 Cloning, Selection and Screening of Llama V_(H)H FragmentsNeutralising Lactococcus lactis Bacteriophage P2

[0061] 2.1 Isolation of V_(H)H Fragments Against Lactoccus lactisBacteriophage P2

[0062] From the llama, positively responding against bacteriophage P2 astested in ELISA, a blood sample of about 200 ml was taken and anenriched lymphocyte population was obtained via centrifugation on aFicoll (Pharmacia) discontinuous gradient. From these cells, total PNAwas isolated by guanidium thiocyanate extraction (e.g. via the methoddescribed by Chomczynnski and Sacchi (1987), Analytical Biochem., 162,156-159. After first strand cDNA synthesis using MMLV-RT (Gibco-BRL) andrandom oligonucleotide primers (Pharmacia), DNA fragments encodingV_(H)H fragments and part of the long or short hinge region wereamplified by PCR using specific primers:            PstI V_(H)-2B5′-AGGTSMARCTGCAGSAGTCWGG-3′ (SEQ. ID NO. 4) S = C and G, M = A and C, R= A and G, W = A and T,           HindIII Lam-07 5′-AACAGTTAAGCTTCCGCTTGCGGCCGCGGAGCTGGGGTCTTCGCTGTGGTGCG-3′ (SEQ. ID NO. 5)(short hinge)           HindIII Lam-085′-AACAGTTAAGCTTCCGCTTGCGGCCGCTGGTTGTGGTTTTGGTGTCTTGGGTT-3′ (SEQ. ID NO.6) (long hinge)

[0063] The DNA-fragments generated by PCR were digested with PstI(coinciding with codon 4 and 5 of the V_(H)H domain, encoding the aminoacids L-Q) and HindIII (introduced at the 5′ end of the hinge specificoligonucleotide primers, coinciding with the amino acid sequence S-L-T),and cloned in the phagemid vector pUR476 (identical to pHEN1 (Hoogenboomet al, Nucleic Acids Res., (1990), 19, 4133-4137) containing the lacIelement as described by Orum et al, Nucleic Acid Res., (1993), 21,4491-4498) as gene-fragments encoding the V_(H)H-domain including thehinge region fused to the geneIII protein of the E. coli bacteriophageM13, thereby enabling display of the antibody fragment on the surface ofthe filamentous phage (McCafferty et al (1990), Nature, 6, 552-554).

[0064] 2.2 Enrichment of Lactococcus Bacteriophage Binding V_(H)HDomains via Phage Display Methodology

[0065] I) A display library with 1×10⁷ clones, of which 75% contained acomplete V_(H)H encoding insert, was constructed in phagemid vectorpUR4676. Phage particles exposing V_(H)H fragments were prepared byinfection of E. coli cells harbouring the phagemid with helperphageVCS-M13 (Marks et al (1991), J. Mol. Biol., 222, 581-597). Byprecipitation of phage from the culture supernatant with PEG6000, freeV_(H)H fragments were removed, thereby avoiding a disturbing competitionfor binding to antigen between phage bound and free V_(H)H domains.

[0066] II) Phage antibodies binding to lactococcus bacteriophage P2,immobilised on maxisorp immunotubes, were selected from the library viathe biopanning procedure (McCafferty et al (1990), Nature, 6, 552-554).After an extensive washing procedure, E. coli phage was eluted from thetube with 0.1 M triethylamine (Baker) by disruption of theantigen-antibody binding with this alkaline shock. After neutralisationwith 0.5 volume of 1 M Tris-HCl pH 7.4, phage was rescued bytransfection into the E. coli host TG1. A renewed selection wasperformed with phage prepared from the transfected population of E. colibacteria as was described before.

[0067] Alternatively, ‘in solution’ capture of E. coli phage exposinglactococcus phage specific antibody fragments was performed with invitro biotinylated bacteriophage P2. Antigen-antibody complexes andassociated phage particles were pulled out of the solution withstreptavidin coated magnetic beads (Dynal) (see Hawkins et al (1992) J.Mol. Biol., 226, 889-896). After washing, E. coli phage was eluted withtriethylamine as described before.

[0068] Individual E. coli clones obtained after the two rounds ofselection were grown in wells of microtiter plates, and the productionof V_(H)H fragments was induced by the addition ofisopropyl-β-D-thiogalactopyranoside (IPTG, 0.1 mM). After 16 hours ofgrowth, the culture supernatant of the clones was analysed in ELISA forthe presence of V_(H)H fragments, which specifically bind to immobilisedbacteriophage P2. Bound V_(H)H fragments were detected with rabbitanti-llama V_(H)H polyclonal antibodies followed by incubation with goatanti-rabbit polyclonal antibodies conjugated to horse radish peroxidase(BIORAD), or with mouse monoclonal anti-myc antibody followed byincubation with polyclonal rabbit-anti-mouse conjugated to horse radishperoxidase (DAKO).

[0069] 2.2.1 Alternative Enrichment Method

[0070] Following the method of Example 2.2(I) above, a library of phagebound V_(H)H domains may be prepared. After incubation of the E. coliphage with in vitro biotinylated lactococcus bacteriophage P2 for twohours, E. coli phage clones exposing non-neutralising, but phage P2specific VHH fragments may be captured with an excess of host cells fromstrain L. lactis. The E. coli phage particles complexed to biotinylatedphage P2 via their exposed VHH fragments, but which are not bound to L.lactis (and thereby potentially neutralising), may be captured fromsolution with virus specific ligands such as streptavidin coatedmagnetic beads, and thus separated from E. coli phage not bound viatheir exposed VHH fragment to phage P2. After elution with a pH-shock(0.1 M triethylamine), the phage population enriched for neutralisingVHH domains may be rescued by infection of E. coli host cells.

[0071] As an alternative method, unlabeled phage P2 can be used insteadof biotinylated phage for binding to VHH-exposed E. coli phage. Aftercapture of clones exposing non-neutralising VHH fragments with L.lactis, a population of clones displaying neutralising binding domainproteins can be captured from solution with monoclonal or polyclonalantibodies directed against L. lactis bacteriophage P2, which wereimmobilised on a solid surface or coupled to a matrix.

[0072] Individual E. coli clones may be grown in wells of microtiterplates, and the production of VHH fragments induced by addition of IPTG(0.1 mM). Culture supernatants containing free VHH domains may be testedin ELISA for binding to L. lactis bacteriophage P2 using the myc-TAG fordetection and for their inhibiting capacity in the high throughput assayusing the techniques described above.

[0073] 2.3 Development of a High-Throughput Screening Assay for theIdentification of Bacteriophage Neutralising V_(H)H Fragments

[0074] The phage neutralising capacity of the V_(H)H fragments wasdemonstrated by a continued growth of the host cell L. lactis. Asmeasure for cell growth the acidification in milk was followed with theincluded pH indicator bromophenol red, which changes from purple-red (pHis 6.5 to 7.0) at the start of cultivation to yellow (pH 4.5 to 5.0)after 8 to 15 hours of growth. 50 μl supernatant of individual clonesderived from the selections with E. coli or S. cerevisiae was mixed with50 μl phage solution (2*10⁹ pfu/ml diluted in semi-skimmed milksupplemented with 0.35% peptone, 0.35% yeast extract, 1% glucose, 0.8%Polymixin B) in a well of a microtiter plate. Subsequently, 100 μl of L.lactis cells (50-fold diluted overnight culture in semi-skimmed milkmedium ascribed before, supplemented with 2% bromophenol red). After 8to 15 hours of incubation at 30° C., ten neutralising antibodies out of285 analysed V_(H)H fragments were identified by the change in colour(yellow). Three of these were characterised in detail (see section 2.6and further).

[0075] 2.4 Sequences of Bacteriophage Neutralising V_(H)H Fragments

[0076] As indicated in the preceding paragraphs anti-LAB-phage V_(H)Hfragments were obtained, which are capable to neutralise lactococcusbacteriophage P2. The sequences of three of such fragments are presentedbelow:

[0077] VHH#1

[0078] (cloned in E. coli phagemid vector pUR3827 and in S. cerevisiaeepisomal plasmid pUR3834): QVQLQESGGG LVQAGGSLRL SCTASRRTGS NWCMGWFRQLAGKEPELVVA LNFDYDMTYY (SEQ. ID NO. 1) ADSVKGRFTV SRDSGKNTVY LQMNSLKPEDTAIYYCAARS GGFSSNRELY DGWGQGTQVT VSS

[0079] VHH#2

[0080] (in E. coli plasmid pUR3828 and in S. cerevisiae episomal plasmidpUR3835): QVQLQESGGG LVQAGGSLRL SCTASRRTGS NWSMGWKRQL AGKEREFVVALNLDYDIPYY (SEQ. ID NO. 2) ADSVKGRFTV STDSGKNTVY LQMNSLKPED TAIYFCAARSGGFSSNRTYY DYWGQGTQVT VSS

[0081] VHH#3

[0082] (in E. coli plasmid pUR3829 and in S. cerevisiae episomal plasmidpUR3836): QVQLQQSGGG LVQRGGSLRL SCTASRRTGS NWSMGWFRQF AGKEPDLLVALNLDYDVPYY (SEQ. ID NO. 3) ADSVKGRFTV SGDSGKNTVY LQMNNLKPED TAIYYCAARSGGFSSNRALY DGWGQGTQVT VSS

Example 3 The Efficiency of V_(H)H Fragments in Neutralising Lactococcuslactis Bacteriophage P2

[0083] 3.1 Recloning in Episomal Plasmid System for Production of V_(H)HFragments in S. cerevisiae

[0084] The V_(H)H encoding genes of clones VHH#1, VHH#2 and VHH#3 weredigested with PstI (present at the 5′ end of the V_(H)H gene andintroduced by primer VH-2B (SEQ. ID. NO. 1)) and BstEII (naturallyoccurring at the 3′ end of most V_(H)H genes) and BstEII from the E.coli phagemid vectors pUR3827, pUR3828 and pUR3829 respectively, andcloned in the episomal S. cerevisiae secretion plasmid pUR4547, therebyobtaining pUR3834, pUR3835 and pUR3836 respectively. Plasmid pUR4547(deposited as CBS100012), with an Ori for autonomous replication in S.cerevisiae, enables the production via the inducible Gal7 promotor;secretion is accomplished by fusing the SUC leader sequence (Harmsen etal (1993), Gene, 125, 115-123) to the amino terminus of the V_(H)Hproduct. The production was examined by analysis of the median fractionobtained after 48 hours of cultivation at 30° C. from 5 clones of eachconstruct on a Coomassie blue stained polyacrylamide gel.

[0085] Plasmid pUR4547 was deposited under the Budapest Treaty at theCentraal Bureau voor Schimmelcultures, Baarn on Aug. 18, 1997 withdeposition number: CBS 100012. In accordance with Rule 28(4) EPC, or asimilar arrangement from a state not being a contracting state of theEPC, it is hereby requested that a sample of such deposit, whenrequested, will be submitted to an expert only.

[0086] 3.2 Construction of Stable V_(H)H Producing Clones of S.cerevisiae by Multi-Copy Integration in the Genome

[0087] Integration of the genes encoding the antibody fragments forestablishing stable secreting S. cerevisiae cell lines was accomplishedby homologous recombination into the yeast genome. By choosing amulti-copy locus, i.e. the ribosomal DNA-(rDNA) locus containing between100 and 150 rDNA units, the insertion of multiple copies was forced,thereby yielding high production levels of antibody fragment. The V_(H)Hgene of clone #1 was digested with the restriction enzymes SacI (locatedbefore the SUC leader sequence) and HindIII (located behind thestopcodon of the V_(H)H gene) and HindIII from the episomal secretionplasmid pUR3834, and cloned in the integration plasmid pUR2778(Giuseppin et al (1991), WO 91100923; Driedonks et al (1995), Yeast, 11,849-864). This plasmid contains the Gal7 promoter for inducibleexpression of the V_(H)H gene product (without tags for identificationor purification), the selectable markers bla (β-lactamase) todiscriminate transformants in E. coli by resistance to the antibioticumampicillin and Leu2d (β-isopropylmalate dehydrogenase) for propagationof transformed S. cerevisiae, an E. coli origin of replication, andfinally the flanking homologous sequences for recombination into thegenome of S. cerevisiae. Transformants in E. coli containing constructswith the V_(H)H gene were identified by restriction enzyme analysis.Plasmid purified with the Nucleobond AX100 kit was used fortransformation of Saccharomyces cerevisiae strain VWK18gal1::URA3 withthe lithiumacetate procedure (Gietz and Schiestl (1995), Meth. Mol.Cell. Biol., 5, 255-259). At least 10 individual clones were chosen forproduction in 50-ml cultures; the medium fraction with the secretedV_(H)H fragments was analysed on a Coommassie blue stained SDS PAGE gel.The clone producing antibody fragment VHH#1 most efficiently was codedpUR3858 and it was used for production in a 10-L fermentor. The mediumfraction containing the antibody fragment was concentrated byultrafiltration and further purification was accomplished by means ofion-exchange chromatography (Mono-S-sepharose, Pharmacia). The amount ofpurified antibody was determined by an OD280 measurement, the micro BCAmethod, and confirmed by the analysis on a Coomassie stained SDS PAGEgel.

[0088] 3.3 Neutralisation Measured by the Inhibitory Effect on PlaqueFormation

[0089] To test the neutralising effect of the anti-phage P2 V_(H)H, thereduction in the phage titers was determined. Therefore antibodyfragments, produced by S. cerevisiae containing plasmid pUR3834 encodingthe neutralising anti-LAB phage VHH#1, or plasmid pUR3831 encoding theLAB phage binding but non-neutralising VHH#4, or construct pUR3850(PCT/EP98/06991) encoding the neutralising bihead molecule VHH#4-#5,made up of the non-neutralising V_(H)H-fragments VHH#4 and VPtH#5 (ofthe following sequences: VHH#4: QVQLQESGGG LVQPGGSLRL SCVVSGEGFSNYPMGWYRQA PGKQRELVAA (SEQ. ID NO. 7) MSEGGDRTNY ADAVKGRFTI SRDNAKKTVYLQMSSLKPED TAVYYCNAAR WDLGPAPFGS WGQGTQVTVS S VHH#5: QVQLQESGGGLVQPGGSLRL SCAVSGAPFR ESTMAWYRQT PGKERETVAF (SEQ. ID NO. 8) ITSGGSKTYGVSVQGRFTIS RDSDRRTVLL QMNNLQPEDT AVYYCHRALS NTWGQGIQVT VSS

[0090] were purified as described be ore. From she monovalent fragments100 and 5 μg, and 5 and 0.25 μg of the bivalent fragment, were mixedwith 5.0*10⁸ phage P2 in 1 ml total volume (diluted in phage buffer: 20mM Tris-HCl pH 7.4, 100 mM NaCl, 10 MM MgSO₄) and incubated for 0.5hours at 37° C. From this incubation mixture 100 μl undiluted solution,10⁻², 10⁻⁴ and 10⁻⁶ diluted solution was added to 100 μl of a culture ofLactococcus lactis subsp. cremoris LM0230 (1*10⁹ cfu/ml), which wasgrown overnight in M17. After the addition of 3 ml of M17 top-agar, themixture was poured on a plate of M17 containing 0.5% glucose and 10 mMCaCl₂. Plates were incubated overnight at 30° C.

[0091]FIG. 1 shows that at a concentration of 5 μg/ml V_(H)H fragmentVHH#1 gave a reduction of more than 99% in the phage titre relative tothe titre found for the control where no antibody fragment was added tophage. An ELISA positive, lactococcus phage P2 specific V_(H)H fragmentselected from the same antibody library, which was classified asnon-neutralising in the high-throughput screening assay, gave nodetectable level of neutralisation, even at concentrations of 100 μg/ml.The bihead molecule VHH#4-#5 did not inhibit infection, at least whenthe phage 22 was present a such high titres; the example described belowshows that the bihead molecule is effective at lower titres of phage.

[0092] The results demonstrates extremely efficient inhibition(neutralisation) of bacteriophage 22 by the monovalent fragments heredescribed.

[0093] 3.4 The Efficiency of Phage Neutralisation Determined by theAcidification of Milk at 30 ml Scale

[0094] In a further aspect, the acidification of milk upon inoculationwith lactic acid bacteria at 30° C. was followed by the regularmeasurement of the pH. For this purpose 30 ml XVM-glucose medium (skimmilk solution containing 0.35% yeast extract, 0.35% peptone and 1%glucose) was inoculated with 300 μl of an overnight culture (109 cfu/ml)of Lactococcus lactis subsp. cremoris LM0230. Alternatively, strain C2was used, which is the LM0230 derived strain, producing protease, andtherefore these bacteria can grow in skim milk without peptone andglucose. The cultures were incubated for 17 h at 30° C. after additionof variable amounts of purified V_(H)H fragments. The XVM is acidifiedby the culture in a period of 8 hours (FIG. 2). When 10³ pfu/ml P2 phagewas added to the culture of LM0230 or C2 in a parallel experiment, noacidification occurred during the whole period of 17 hours (FIG. 2,panel A and B). Addition of the monovalent antibody fragment VHH#1(pUR3834) to the culture containing phage P2, resulted in a completelyrestored acidification profile (FIG. 2, panel A).

[0095] The bihead molecule VHH#4-#5 also prevented phage infection, butthe neutralizing character stemmed from its bivalency as could beconcluded from experiments with the monovalent fragments VHH#4 andVHH#5, which as separately added fragments did not inhibit (FIG. 2,panel B).

[0096] 3.5 Conformation of the Monovalent Character of the PhageNeutralising V_(H)H Fragments

[0097] In order to exclude possible aggregation of the V_(H)H fragments,which might lead to the formation of dimers or higher orders ofmultimers, as has been observed for single chain antibodies (Holliger etal (1993), Proc. Natl. Acad. Sci., 90, 6444-6448; Kortt et al (1997),Protein Eng., 10, 423-433), the produced molecules were analysed in anELISA based test. In this assay bacteriophage was immobilised (at aconcentration of 10¹⁰ pfu per ml of PBS) and as a detection probe invitro biotinylated bacteriophage was used. Bivalent V_(H)H fragment suchas the bihead construct or polyclonal antibody (FIG. 3 panel B) presentin sera from the immunised llama gave positive responses when incubatedwith biotinylated phage (prepared with NHS-biotin (Pierce) according tothe instruction of the supplier) and horse radish peroxidase labelledstreptavidin (DAKO); the principle of the assay is shown in FIG. 3(panel A). In contrast, the non-neutralising V_(H)H fragments VHH#4 andVHH#5 and the neutralising V_(H)H fragments VHH#1, VHH#2 and VHH#3 aswell as the polyclonal serum, taken from the llama prior to immunisationwith bacteriophage, were not detected with biotinylated phage (FIG. 3,panel B).

[0098] These experiments showed that the produced V_(H)H fragments aremonomeric. Therefore the inhibiting effect is not obtained bycross-linking of bacteriophage particles, but rather is determined bythe epitope(s) recognised by these particular antibodies.

[0099] 3.6 Neutralisation of Other Species of Lactococcus Phages

[0100] Lactococcus phages have been classified into 12 species. Of thoseonly three species have been found to interfere with industrial milkfermentations, i.e. prolate headed c2 species and the isometric-headed936 (most often found in factories) and P335 species. The bacteriophageP2, which was used for immunisation of the llama and selection of theantibody display library, belongs to the 936 species. Therefore theneutralising capacity of VHH#1 was examined against another member(phage SK1) of the 936 species, but also against two members (phage Q38and c2) of the prolate headed c2 species.

[0101] With the microtiter plate assay described in example 2.3 thecross-reactivity and the neutralising capacity was analysed on theacidification of phage infected cultures as measure for phageresistance. To the mixture of host cells (at a density of 10⁶ cfu/ml)and bacteriophage (at a titer of 10³ pfu/ml) variable amounts ofantibody fragment VHH#1 were added; after 15 hours of cultivation at 30°C. the neutralising activity could be observed by the colour change ofthe included indicator bromophenol red. The following combinations weretested: L. lactis strain C2 with phage P2, strain SMQ-196 and phage Q38and bacterial strain LM230 with either phage SK1 or phage c2. Besidesthe described antibody fragment VHH#1 the polyclonal pre- and postimmunesera from the llama were used as negative and positive control as wellas not-infected bacterial host.

[0102] The two tested phages p2 and SK1 of the isometric-headed 936species were effectively neutralised by the monovalent antibody fragment(up to a dilution of 47 ng/ml) and post-immune serum (up to a 10-4 folddilution). In contrast, the two members Q38 and c2 of the prolate-headedc2 species were not inhibited by VHH#1, not even at concentrations of0.47 mg/ml, while some neutralisation was observed with post-immuneserum at a 10-fold dilution.

[0103] By using this limited number of different phage types it wasconcluded that members of the isometric-headed species (used forimmunisation) were neutralised effectively by VHH#1, but that phagebelonging to the distantly related prolate-headed c2 species were notinhibited.

Example 4 Protecting a Cheese Starter Culture from Infection With PhageDuring the Production and the Ripening of Semi-Hard Gouda Cheese

[0104] Gouda-type cheeses were produced on pilot scale (200 L batches ofcheese milk, yielding 4 cheeses of about 6 kg per batch) in open cheesevats. The cheese milk is treated by thermization (68° C., 14 sec) andbactofugation, before being standardised to achieve a cheese fat contentof about 50% (dry matter). The milk subsequently is pasteurised for 10sec at 72° C. To the cheese milk, the following components were added:

[0105] At t=0 min: bacteriophage P2 in different levels (Table I)

[0106] At t=5 min: addition of CaCl₂ (23.1 g/100 l), NaNO₃ (15.5 g/100l) and different levels of the monovalent antibody fragment VHH#1

[0107] At t=10 min: addition of 40 g/100 l starter culture Lactococcuslactis C2 (fully grown in milk)

[0108] At t=15 min: addition of 23 g/100 l calf rennet

[0109] All additions were poured in slowly to the stirred cheese milk toguarantee complete mixing. Further processing (renneting, cutting, curdwashing, scalding, draining and filling) was carried out as usual forstandard Gouda-type cheese, according to well-known processes asdescribed by Kammerlehner (1) and by De Vries and Van Ginkel (2).Brining was started when the cheeses had reached a pH of 5.5 to 5.4.Cheeses were ripened at 13° C. at 88% relative humidity.

[0110] During ripening, samples were taken for the following analysis:

[0111] at 2 weeks for the general chemical analysis

[0112] at 2, 6, 13 weeks for protein degradation (total, soluble andamino acid nitrogen (3))

[0113] at 13 weeks for an extensive aroma analysis

[0114] Samples were stored frozen at −40° C. before analysis. TABLE ICompilation of the 3 separate cheese experiments performed Phage Phagein VHH#1 VHH#1 in pH Code (pfu/ml) whey (μg/ml) whey (6 h; 24 h) 1.1 — —— − 5.40; 5.22 1.2 — — 1 + 5.40; 5.21 1.3 1.0 × 10³ 2.2 × 10³ — − 6.00;5.65 1.4 1.0 × 10³ — 1 + 5.46; 5.19 1.5 1.2 × 10⁴ — 1 + 5.40; 5.26 2.1 —— — − 5.51; 5.16 2.2 1.8 × 10³ —   0.1 + 5.59; 5.16 2.3 1.8 × 10⁴ — 1 +5.57; 5.18 2.4 2.1 × 10⁴ —   0.1 − 5.53; 5.21 2.5 1.7 × 10³ 3.0 × 10³ —− 5.90; 5.58 3.1 — — — − 5.44; 5.22 3.2 1.4 × 10³ —   0.1 + 5.45; 5.203.3 1.3 × 10⁴ — 1 + 5.49; 5.21 3.4 1.5 × 10⁵ — 1 + 5.48; 5.21 3.5 1.8 ×10⁵ 27   0.1 ???? 5.45; 5.24

[0115] The results depicted in Table I clearly show that addition of themonovalent antibody fragment VHH#1 to the P2 phage infected cheese milk,prior to the addition of the starter culture L. lactis C2, projects theculture against phage infection. Phages are not detected anymore in thewhey even if the cheese milk is infected with 1.5×10⁵ pfu/ml, when 1μg/ml of VHH#1 is present. The acidification of the cheese milk is asexpected (cf. 3.1 with 3.4). When the antibody fragment is not added,phages are detected in the whey and the acidification slows downsignificantly (cf. 1.1 and 1.3 or 2.1 and 2.5). When the antibodyfragment is added, the activity still can be found back in the whey,even if the phage is added prior to the antibody. This proves that theantibody is present in excess and is able to neutralise the phagecompletely. As can be seen from experiment 3.5, the limit is possiblyreached when 0.1 μg/ml VHH#1 is added to cheese milk infected with1.8×10⁵ pfu/ml. This level of phage infection is considered to beextremely high in a normal operating cheese plant.

[0116] To determine if the neutralisation of the phage during theinitial cheese fermentation is enough to obtain a normal cheese ripeningprocess, the chemical composition of the cheeses has been determinedafter 2 weeks as well as the proteolysis after 2, 6 and 13 weeks (TableII and III). TABLE II Chemical composition cheeses after 2 weeks % Fat %Salt Analysis % Humidity % Fat i.d.s. % Salt i.d.s. pH 1.1 41.4 29.650.4 1.9 3.1 5.17 1.2 41.6 29.4 50.3 1.9 3.2 5.16 1.3 40.9 29.9 50.4 2.33.9 5.63 1.4 40.1 29.6 50.2 1.9 3.3 5.18 1.5 41.5 29.9 51.0 1.8 3.1 5.142.1 42.5 29.2 50.9 2.2 3.8 5.13 2.2 41.2 29.7 50.6 2.2 3.7 5.14 2.3 41.230.0 51.0 2.2 3.7 5.15 2.4 39.7 30.9 51.0 1.8 3.0 5.19 2.5 39.6 31.251.4 1.8 3.0 5.60 3.1 42.3 30.0 51.7 1.7 3.0 5.16 3.2 41.7 30.3 51.8 1.72.9 5.17 3.3 41.4 30.2 51.4 1.6 2.8 5.19 3.4 40.4 30.5 51.0 1.5 2.6 5.223.5 40.4 31.1 51.8 1.8 2.8 5.26

[0117] TABLE III Proteolysis during ripening of the cheeses (SolubleNitrogen/Total Nitrogen; Amino acid Nitrogen/TN; AN/SN) Weeks SN/TNAN/TN AN/SN 2 6 13 2 6 13 2 6 13 1.1 7.70 11.80 17.19 1.39 2.13 2.7818.06 18.08 16.19 1.2 7.86 12.31 17.47 1.53 2.23 2.90 19.50 18.14 16.621.3 7.36 11.33 15.33 2.61 4.25 5.79 35.42 37.53 37.76 1.4 7.60 12.3419.32 2.15 2.47 3.39 28.32 20.0 17.55 1.5 7.81 12.94 17.76 1.63 2.654.11 20.92 20.48 23.17 2.1 6.77 10.71 14.96 1.94 2.02 2.75 28.69 18.8718.39 2.2 6.75 11.48 16.14 1.65 2.10 3.37 24.48 18.33 20.90 2.3 6.1111.95 16.72 1.84 2.93 3.83 30.13 24.50 22.90 2.4 6.72 11.42 17.15 2.002.28 3.60 29.77 19.96 20.98 2.5 7.53 12.01 16.92 2.93 4.30 6.16 38.8735.76 36.44 3.1 8.33 12.49 17.28 1.13 1.86 2.65 13.51 14.86 15.36 3.27.72 12.60 17.51 1.33 2.09 3.10 17.27 16.63 17.68 3.3 8.25 12.33 17.751.47 2.22 3.81 17.78 18.05 21.05 3.4 8.21 12.90 18.95 1.58 2.75 4.4219.20 21.32 23.33 3.5 7.96 12.39 18.34 1.49 2.59 4.34 18.77 20.93 23.68

[0118] From the data in Table II it can be concluded that the chemicalcomposition of a cheese is not much influenced by the presence of aphage apart from the acidification (cf. 1.3 and 2.5 with the otherdata). Consequently an additional ripening period of 2 weeks, does notrestore the acidification capacity of the starter culture upon infectionwith the phage, unless the neutralising antibody VHH#1 is added prior tothe starter culture. The data from Table III show that proteolysis, thatis one of the major indicative parameters for cheese ripening, isabnormal in phage infected cheeses and that this once again can benormalised if VHH#1 is added prior to the addition of the starterculture (cf. The AN/TN and AN/SN data of 1.3 and 2.5 with the otherdata). Too many amino acids are liberated when the phage is notneutralised by VHH#1, indicating an unbalanced proteolysis and thereforeand off-flavoured cheese.

REFERENCES

[0119] 1. Kammerlehrer, J. (1989) Lab-Käse Technologie. Band III, p642-643. In Molkereitechnik Band 84/85. Verlag Th. Mann,Gelsenkircher-Buer, ISBN 3-7862-0083-1

[0120] 2. De. Vries E. and van Ginkel, W. (1980) Test of a curd-makingtank. Type “Damrow Double O” with a capacity of 16000 L, manufactured byDEC. NIZO Rapport R113

[0121] 3. Noomen, A., (1977) Noordhollandse Meshanger Cheese: a modelfor research on cheese ripening.2. The ripening of the cheese. Neth.Milk Diary J. 31, 75-102

1 8 1 123 PRT LLAMA 1 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu ValGln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Arg ArgThr Gly Ser Asn Trp 20 25 30 Cys Met Gly Trp Phe Arg Gln Leu Ala Gly LysGlu Pro Glu Leu Val 35 40 45 Val Ala Leu Asn Phe Asp Tyr Asp Met Thr TyrTyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Ser GlyLys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu AspThr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Arg Ser Gly Gly Phe Ser Ser AsnArg Glu Leu Tyr Asp Gly 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr ValSer Ser 115 120 2 123 PRT LLAMA 2 Gln Val Gln Leu Gln Glu Ser Gly GlyGly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr AlaSer Arg Arg Thr Gly Ser Asn Trp 20 25 30 Ser Met Gly Trp Phe Arg Gln LeuAla Gly Lys Glu Arg Glu Phe Val 35 40 45 Val Ala Leu Asn Leu Asp Tyr AspIle Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser ThrAsp Ser Gly Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu LysPro Glu Asp Thr Ala Ile Tyr Phe Cys 85 90 95 Ala Ala Arg Ser Gly Gly PheSer Ser Asn Arg Thr Tyr Tyr Asp Tyr 100 105 110 Trp Gly Gln Gly Thr GlnVal Thr Val Ser Ser 115 120 3 123 PRT LLAMA 3 Gln Val Gln Leu Gln GlnSer Gly Gly Gly Leu Val Gln Arg Gly Gly 1 5 10 15 Ser Leu Arg Leu SerCys Thr Ala Ser Arg Arg Thr Gly Ser Asn Trp 20 25 30 Ser Met Gly Trp PheArg Gln Phe Ala Gly Lys Glu Pro Asp Leu Leu 35 40 45 Val Ala Leu Asn LeuAsp Tyr Asp Val Pro Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe ThrVal Ser Gly Asp Ser Gly Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met AsnAsn Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Arg SerGly Gly Phe Ser Ser Asn Arg Ala Leu Tyr Asp Gly 100 105 110 Trp Gly GlnGly Thr Gln Val Thr Val Ser Ser 115 120 4 22 DNA Artificial SequenceDescription of Artificial SequencePRIMER 4 aggtsmarct gcagsagtcw gg 22 553 DNA Artificial Sequence Description of Artificial SequencePRIMER 5aacagttaag cttccgcttg cggccgcgga gctggggtct tcgctgtggt gcg 53 6 53 DNAArtificial Sequence Description of Artificial SequencePRIMER 6aacagttaag cttccgcttg cggccgctgg ttgtggtttt ggtgtcttgg gtt 53 7 121 PRTLLAMA 7 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Glu Gly Phe Ser AsnTyr 20 25 30 Pro Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu LeuVal 35 40 45 Ala Ala Met Ser Glu Gly Gly Asp Arg Thr Asn Tyr Ala Asp AlaVal 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr ValTyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val TyrTyr Cys 85 90 95 Asn Ala Ala Arg Trp Asp Leu Gly Pro Ala Pro Phe Gly SerTrp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 8 113PRT LLAMA 8 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ala Pro Phe ArgGlu Ser 20 25 30 Thr Met Ala Trp Tyr Arg Gln Thr Pro Gly Lys Glu Arg GluThr Val 35 40 45 Ala Phe Ile Thr Ser Gly Gly Ser Lys Thr Tyr Gly Val SerVal Gln 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Ser Asp Arg Arg Thr ValLeu Leu 65 70 75 80 Gln Met Asn Asn Leu Gln Pro Glu Asp Thr Ala Val TyrTyr Cys His 85 90 95 Arg Ala Leu Ser Asn Thr Trp Gly Gln Gly Ile Gln ValThr Val Ser 100 105 110 Ser

1. A method of inhibiting viral infection using a monovalent antigenbinding protein comprising a single variable domain binding unit, or afunctional equivalent thereof, capable of binding to a virus.
 2. Amethod according to claim 1 wherein the single domain variable domainbinding unit comprises a heavy chain variable domain derived from animmunoglobulin devoid of light chains, or a functional equivalentthereof.
 3. A method according to claim 2 wherein the single variabledomain binding unit comprises a heavy chain variable domain derived froma Camelid immunoglobulin or a functional equivalent thereof.
 4. A methodaccording to claim 1 wherein the virus is a lactococcal bacteriophage.5. A method according to claim 1 wherein the virus is pathogenic tohumans or animals.
 6. A method according claim 1 wherein the virus ispathogenic to plants.
 7. Use of a monovalent antigen binding proteincomprising a single variable domain binding unit, or a functionalequivalent thereof, capable of binding to a virus in inhibiting viralinfection.
 8. Use of a monovalent antigen binding protein comprising asingle variable domain binding unit, or a functional equivalent thereof,capable of binding to a virus in the preparation of a medicament forinhibiting viral infection.
 9. A food product comprising a singlevariable domain binding unit, or a functional equivalent thereof,capable of binding to a virus.
 10. A pharmaceutical or cosmeticcomposition comprising a single variable domain binding unit, or afunctional equivalent thereof, capable of binding to a virus.
 11. Amonovalent antigen binding protein capable of inhibiting viral infectioncomprising a heavy chain variable domain comprising an amino acidsequence as shown in SEQ. ID No. 1, 2 or
 3. 12. Nucleotide sequencescoding for a protein according to claim
 11. 13. Expression vectorcomprising a nucleotide sequence according to claim
 12. 14. A host celltransformed with a vector according to claim
 13. 15. Method forselecting an antigen binding protein capable of inhibiting viralinfection of a host cell comprising the steps of: i) complexing anantigen binding protein with a target virus, ii) exposing the antigenbinding protein-virus complex of step (i) to an excess of host cells,iii) removing the host cells and any associated antigen bindingprotein-virus complex, iv) capturing antigen binding protein-viruscomplex not taken up by the host cells in step (ii) with virus specificligands to separate virus specific antigen binding proteins fromnon-binding proteins.
 16. Method for identifying an antigen bindingprotein capable of inhibiting bacteriophage infection of a lactic acidbacterial cell host comprising the steps of: i) culturing of bacterialhost cells in the presence of antigen binding protein and bacteriophage,ii) assaying said culture for active cell growth manifest in a change inpH of the culture growth medium.